1Department of Horticultural Science, Gyeongnam National University of Science and Technology (GNTech), Jinju 52725, Korea, 2College of Applied Life Science, Faculty of Bioscience and Industry, The Research Institute for Subtropical Agriculture and Biotechnology, Jeju National University, Jeju 63243, Korea

Received June 30, 2017; Revised August 16, 2017; Accepted August 21, 2017.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The objective of this study was to determine inhibitory activities of four volatile plant essential oils (cinnamon oil, fennel oil, origanum oil and thyme oil) on in vitro growth of Fusarium oxysporum f. sp. fragariae causing Fusarium wilt of strawberry plants. Results showed that these essential oils inhibited in vitro conidial germination and mycelial growth of F. oxysporum f. sp. fragariae in a dose-dependent manner. Cinnamon oil was found to be most effective one in suppressing conidial germination while fennel oil, origanum oil and thyme oil showed moderate inhibition of conidial germination at similar levels. Cinnamon oil, origanum oil and thyme oil showed moderate antifungal activities against mycelial growth at similar levels while fennel oil had relatively lower antifungal activity against mycelial growth. Antifungal effects of these four plant essential oils in different combinations on in vitro fungal growth were also evaluated. These essential oils demonstrated synergistic antifungal activities against conidial germination and mycelial growth of F. oxysporum f. sp. fragariae in vitro. Simultaneous application of origanum oil and thyme oil enhanced their antimicrobial activities against conidial germination and fungal mycelial growth. These results underpin that volatile plant essential oils could be used in eco-friendly integrated disease management of Fusarium wilt in strawberry fields.

Volatile antifungal activities of these four plant essential oils against in vitro conidial germination and mycelial growth were evaluated using method described in our previous study (Hong et al., 2015) with slight modifications. Briefly, for conidial germination-inhibiting assays, four conidial suspension drops (20 μl each) prepared in 1/4 PDB on glass slides were treated with plant essential oils for 10 h at 25°C in plastic square dishes (100 mm × 100 mm × 13 mm). Conidia were stained with 1 μl of lactophenol-trypan blue solution and conidial suspensions were covered by coverslips before observation under a microscope. For mycelial growth-inhibiting assays, fungal mycelial plugs (5 mm in diameter) were inoculated onto the centre of 10 ml of PDA media in Petri dishes (85 mm in diameter, 13 mm in height). Fungal colony diameters were measured at 6 days after inoculation onto PDA media at 25°C. Diameters of fungal colonies treated with plant essential oils were compared to those of untreated colonies and expressed as percentage (%).

Simultaneous treatment with different plant essential oils

Different plant essential oils were applied simultaneously to determine their synergistic efficacies against in vitro conidial germination and mycelial growth of F. oxysporum f. sp. fragariae. Paper discs containing 0.5 and 1 μl/disc of each plant essential oil were attached to the inside plate cover singly or concurrently with other paper disc(s) for conidial germination and mycelial growth inhibition assays, respectively. Conidial germination and mycelial growth were evaluated at 10 h and 6 days after incubation at 25°C with different combinations of these plant essential oils described above.

Different antifungal efficacies of four plant essential oils against in vitro conidial germination of F. oxysporum f. sp. fragariae

The four plant essential oils tested in this study showed different antifungal efficacies against in vitro conidial germination of F. oxysporum f. sp. fragariae in dose-dependent manners (Fig. 1). Treatment with cinnamon oil drastically suppressed conidial germination at the lowest dose used (0.1 μl/disc). Conidial germination was completely inhibited by cinnamon oil at 0.2 μl/disc. Fennel oil was less effective in reducing conidial germination compared to cinnamon oil. It failed to completely suppress conidial germination even at the highest dose used in this study (5 μl/disc). Origanum oil and thyme oil demonstrated moderate antifungal activities against conidial germination at similar levels. Both of them inhibited conidial germination in a dose-dependent manner. Increasing concentrations of origanum oil and thyme oil concentrations resulted in gradual decreases of conidial germination. No germination was found in conidia treated with 5 μl/disc of thyme oil.

Different antifungal efficacies of four plant essential oils against in vitro mycelial growth of F. oxysporum f. sp. fragariae

Different combinations of these four plant essential oils were applied to investigate whether they might have synergism in suppressing conidial germination and mycelial growth of F. oxysporum f. sp. fragariae (Fig. 3, 4). Several combinations of these plant essential oils synergistically suppressed conidial germination (Fig. 3A). Treatment with cinnamon oil alone almost completely suppressed conidial germination. Additional treatment with other plant essential oils did not significantly increase the high suppressive activity of cinnamon oil. Three different combinations of two different essential oils (fennel oil + origanum oil, fennel oil + thyme oil, origanum oil + thyme oil) significantly increased their antifungal activities against conidial germination compared to any single plant essential oil treatment. No additional increase in antifungal activity against conidial germination was found by mixing three or four plant essential oils (Fig. 3B).

Enhanced suppressive activities against fungal mycelial growth were demonstrated by combined treatments with different plant essential oils (Fig. 4). After treatments with different combinations of essential oils, colony formation on PDA was demonstrated at 6 days after culture (Fig. 4A). Cinnamon oil treatment alone slightly reduced mycelial growth. Additional application with fennel oil failed to enhance its antifungal activity. However, addition application with origanum oil or thyme oil accelerated mycelial growth restriction caused by cinnamon oil. Single treatment with origanum oil or thyme oil drastically suppressed mycelial growth. Combined treatment with fennel oil and origanum oil showed synergistic effect on the suppression of mycelial growth compared to single treatment. Triple treatment with cinnamon oil, fennel oil and origanum oil resulted in more reduction of mycelial growth. Antifungal activity against mycelial growths by triple treatment with cinnamon oil, origanum oil and thyme oil was similar to that by dual treatment with origanum oil and thyme oil. Triple treatment with cinnamon oil, fennel oil and thyme oil exerted additive antifungal activity compared to treatment with a combination of any two plant essential oils. Fennel oil treatment did not enhance the antifungal activity of dual treatment with origanum oil and thyme oil. Antifungal activity with a combination of all four essential oils was not significantly different from that with a combination of origanum and thyme oil (Fig. 4B).

Discussion

Due to their antifungal activities, plant essential oils have been applied in eco-friendly postharvest management to extend transportation and storage periods as well as shelf life of strawberry fruits in markets (Bhaskara Reddy et al., 1998; Nabigol and Morshedi, 2011). Several fungal species such as Botrytis cinerea, Rhizopus stolonifer and Aspergillus niger frequently deteriorating strawberry fruit quality have been controlled by various plant essential oils. However, control method to mitigate Fusarium wilt occurrence in strawberry plant culture fields is limited in spite of growing concerns of its worldwide spread. Antifungal activities of several plant essential oils against F. oxysporum causing Fusarium wilt in a variety of crop species have been evaluated. Clove oil has been found to be more effective in inhibiting in vitro conidial germination and mycelial growth of F. oxysporum f. sp. lycopersici causing tomato Fusarium wilt, compared to essential oils from lemongrass, mint, or eucalyptus plants (Sharma et al., 2017). Tomato Fusarium wilt disease has been efficiently reduced by treating soil mixtures with aqueous emulsion of clove oil (La Torre et al., 2016; Sharma et al., 2017). In this study, different antifungal activities of four plant essential oils against strawberry fungus F. oxysporum f. sp. fragariae were demonstrated in vitro. Our results suggest that these essential oils might be useful to reduce Fusarium wilt in strawberry growing fields.

These four plant essential oils (cinnamon oil, fennel oil, origanum oil and thyme oil) tested in this study showed different antifungal efficacies against in vitro conidial germination and mycelial growth of F. oxysporum f. sp. fragariae. Cinnamon oil was found to be the most effective one in suppressing conidial germination of F. oxysporum f. sp. fragariae among these four plant essential oils. Origanum oil and thyme oil were more effective in reducing mycelial growth compared to the other two essential oils. These results suggest that various plant essential oils have different antifungal activities against conidial germination and mycelial growth. Cinnamon oil has been widely used to inhibit the growth of various phytopathogenic fungi. It has been reported that conidial germination and mycelial growth of F. oxysporum f. sp. cubense can be significantly suppressed by cinnamon oil, resulting in decreased panama disease in banana seedlings (Monteiro et al., 2013). Therefore, cinnamon oil treatment might be useful to halt early development of Fusarium wilt in strawberry fields via inhibiting conidial germination in soil environments.

Commercial products containing different plant essential oils as a component have been available for crop protection. It has been reported that combined treatment with rosemary oil, clove oil and thyme oil can reduce tomato Fusarium wilt much more efficiently in comparison with control effect with single treatment by clove oil, thyme oil, or rosemary oil (La Torre et al., 2016). This provides evidence that different plant essential oils can be used simultaneously to enhance their antifungal activity and crop protection efficacies. In the present study, combined treatment with origanum oil and thyme oil (1:1, vol/vol) resulted in the best synergistic effect against mycelial growth of F. oxysporum f. sp. fragariae than other combinations. However, antifungal activities of origanum oil and thyme oil mixture at different ratios need to be elucidated to increase their antifungal efficacy.

To apply plant essential oils into crop fields practically, side effects including phytotoxicity should be considered. It has been shown that excess cinnamon oil can result in severe tissue damage, leading to detached tomato leaves (Lee et al., 2012). Dose-dependent phytotoxicities of soil-drenching clove oil to cucumber, muskmelon, pepper and tomato seedlings before transplanting have also been reported (Meyer et al., 2008). Therefore, phytotoxic effect of plant essential oils should be defined before applying them in strawberry fields regarding their dosage, frequency and application method as well as plant cultivar, age and organs.

In summary, our results revealed that the four plant essential oils tested in this study could efficiently suppress conidial germination and mycelial growth of F. oxysporum f. sp. fragariae in vitro. These effects were more evident when two plant essential oils were mixed together compared to single plant essential oil treatment. Therefore, developing commercial products containing several plant essential oils might be promising as eco-friendly strategy to control strawberry Fusarium wilt.

Acknowledgments

This research was supported by Gyeongnam National University of Science and Technology (GNTech) Grant 2016 to J. K. H.

Figures

Fig. 1. Inhibition of conidial germination of Fusarium oxysporum f. sp. fragariae by four different volatile plant essential oils (cinnamon oil, fennel oil, origanum oil and thyme oil). Germinated conidia of F. oxysporum f. sp. fragariae SFW-10 on glass slides after treatment with different doses (0, 0.1, 0.2, 0.5, 1, 2 and 5 μl/disc) of each volatile essential oil were observed under a light microscope. Relative conidial germination (%) inhibited by different doses of each essential oil was shown as percentage of conidial germination compared to control. Error bars represent standard errors of means of four independent experimental replications. Means followed by the same letter are not significantly different at 5% level by least significant difference test. The same letter above bars represented no significant difference between treatments.

Fig. 2. Mycelial growth of Fusarium oxysporum f. sp. fragariae inhibited by four volatile plant essential oils (cinnamon oil, fennel oil, origanum oil and thyme oil). Colony formation of F. oxysporum f. sp. fragariae SFW-10 on PDA media after treatment with different doses (0, 0.1, 0.2, 0.5, 1, 2 and 5 μl/disc) of each volatile essential oil was determined. Relative mycelial growth inhibited by different doses of each essential oil is shown as percentage of fungal colonies on PDA. Error bars represent standard errors of means of four independent experimental replications. Means followed by the same letter are not significantly different at 5% level by least significant difference test. The same letter above bars represented no significant difference between treatments.

Fig. 3. Combination effect of four plant essential oils (cinnamon oil (C), fennel oil (F), origanum oil (O) and thyme oil (T)) on their inhibitory activities against conidial germination of Fusarium oxysporum f. sp. fragariae. (A) Conidial germination at 10 h after incubation with or without plant essential oil treatment. Germinated conidia of F. oxysporum f. sp. fragariae SFW-10 on glass slides after treatment with 0.5 μl/disc of each volatile essential oil were observed under a light microscope. (B) Relative conidial germination (%) inhibited by different combinations of plant essential oils was shown as percentage of conidial germination compared to control. Error bars represent standard errors of means of four independent experimental replications. Means followed by the same letter are not significantly different at 5% level by least significant difference test. The same letter above bars represented no significant difference between treatments.

Fig. 4. Combination effect of four plant essential oils (cinnamon oil (C), fennel oil (F), origanum oil (O) and thyme oil (T)) on their inhibitory activities against mycelial growth of Fusarium oxysporum f. sp. fragariae. (A) Colony formation of F. oxysporum f. sp. fragariae SFW-10 on PDA media after treatment with 1 μl/disc of each volatile essential oil. Photos were taken at 6 days after culture at 25°C. (B) Relative mycelial growth inhibited by different combinations of plant essential oils shown as percentage of fungal colonies on PDA. Error bars represent standard errors of means of four independent experimental replications. Means followed by the same letter are not significantly different at 5% level by least significant difference test. The same letter above bars represented no significant difference between treatments.